Atrazine-Induced Hermaphroditism By: Michelle Lin

Atrazine-Induced Atrazine-Induced Hermaphroditism Hermaphroditism

By: Michelle Lin By: Michelle Lin

What is Atrazine?What is Atrazine?


Systematic herbicide that blocks photosynthesisSystematic herbicide that blocks photosynthesis


Systematic herbicide that blocks photosynthesisSystematic herbicide that blocks photosynthesis Metabolized to four hydroxyatrazine compounds Metabolized to four hydroxyatrazine compounds

& to three chlorinated atrazine compounds.& to three chlorinated atrazine compounds.



& to three chlorinated atrazine compounds.& to three chlorinated atrazine compounds. Predominate metabolites found in plantsPredominate metabolites found in plants



& to three chlorinated atrazine compounds.& to three chlorinated atrazine compounds. Predominate metabolites found in plantsPredominate metabolites found in plants Desethylated atrazine, desisopropyl atrazine, & Desethylated atrazine, desisopropyl atrazine, &

diaminochlorotriazine (DACT) predominate in diaminochlorotriazine (DACT) predominate in animal tissues, & in soils and wateranimal tissues, & in soils and water





commonly detected pesticide in ground and surface water





commonly detected pesticide in ground and surface water

detected in streams, rivers, groundwater, and reservoirs is related directly to both its volume of usage, and its tendency to persist in soils and move with water.

It’s a pesticide, so what?It’s a pesticide, so what?


Most widely used herbicide in the worldMost widely used herbicide in the world


Most widely used herbicide in the worldMost widely used herbicide in the world Most common contaminant in ground and surface Most common contaminant in ground and surface

waterswaters



waterswaters About 64 to 75 million lbs of active ingredient are About 64 to 75 million lbs of active ingredient are

applied per yearapplied per year




applied per yearapplied per year Three-fourths of all field corn & sorghum are Three-fourths of all field corn & sorghum are

treated with atrazine annually for weed control treated with atrazine annually for weed control




applied per yearapplied per year Three-fourths of all field corn & sorghum are Three-fourths of all field corn & sorghum are

treated with atrazine annually for weed control treated with atrazine annually for weed control Also used on sugarcane, wheat, guava, Also used on sugarcane, wheat, guava,

macadamia nuts orchard grass and haymacadamia nuts orchard grass and hay

What does Atrazine cause?What does Atrazine cause?


Hermaphroditism in American Leopard Frogs Hermaphroditism in American Leopard Frogs ((Rana pipiensRana pipiens))


Hermaphroditism in American Leopard Frogs Hermaphroditism in American Leopard Frogs ((Rana pipiensRana pipiens)) 21 ppb atrazine exposure in 48 hrs can results 21 ppb atrazine exposure in 48 hrs can results

in severe gonadal dygenesis in African clawed in severe gonadal dygenesis in African clawed frogs (frogs (Xenopus laevis)Xenopus laevis)


Hermaphroditism in American Leopard Frogs Hermaphroditism in American Leopard Frogs ((Rana pipiensRana pipiens)) 21 ppb atrazine exposure in 48 hrs can results 21 ppb atrazine exposure in 48 hrs can results

in severe gonadal dygenesis in African clawed in severe gonadal dygenesis in African clawed frogs (frogs (Xenopus laevis)Xenopus laevis)

at concentrations of 0.1 ppb, atrazine induces at concentrations of 0.1 ppb, atrazine induces hermaphroditism hermaphroditism

The experimentThe experiment


Leopard frogs obtained from Sensiba March, Leopard frogs obtained from Sensiba March, Brown County, Wisconsin then shipped to the Brown County, Wisconsin then shipped to the University of California BerkeleyUniversity of California Berkeley



Larvae were treated by immersion with nominal Larvae were treated by immersion with nominal concentrations of 0, 0.1, or 25 ppb atrazine (98% concentrations of 0, 0.1, or 25 ppb atrazine (98% pure) pure)




Atrazine was predissolved in ethanol, and all Atrazine was predissolved in ethanol, and all treatments contained 0.0036% ethanol treatments contained 0.0036% ethanol





Exposed throughout larval period from 2 days Exposed throughout larval period from 2 days posthatching until complete tail reabsorbtion.posthatching until complete tail reabsorbtion.


Leopard frogs obtained from Sensiba March, Brown Leopard frogs obtained from Sensiba March, Brown County, Wisconsin then shipped to the University County, Wisconsin then shipped to the University of California Berkeleyof California Berkeley



Exposed throughout larval period from 2 days Exposed throughout larval period from 2 days posthatching until complete tail reabsorbtion.posthatching until complete tail reabsorbtion.

All dosing and analyses were conducted blindly All dosing and analyses were conducted blindly with color-coded tanks and treatments. with color-coded tanks and treatments.

The experiment continuedThe experiment continued


At metamorphosis, each animal was weighed and At metamorphosis, each animal was weighed and measured then euthanized in 0.2% benzocaine, measured then euthanized in 0.2% benzocaine, fixed in Bouin’s fixative, and preserved in 70% fixed in Bouin’s fixative, and preserved in 70% ethanol until further analysis.ethanol until further analysis.



Sex of all individuals were determined based on Sex of all individuals were determined based on gross gonadal morphology using a Nikon SMZ 10Agross gonadal morphology using a Nikon SMZ 10A




Tissues dissected and dehydrated in graded Tissues dissected and dehydrated in graded alcohols followed by infiltration with Histoclear alcohols followed by infiltration with Histoclear and paraffinand paraffin




Tissues dissected and dehydrated in graded Tissues dissected and dehydrated in graded alcohols followed by infiltration with Histoclear alcohols followed by infiltration with Histoclear and paraffinand paraffin

Serial histological sections cut at 8µm through Serial histological sections cut at 8µm through entire gonadentire gonad


At metamorphosis, each animal was weighed and At metamorphosis, each animal was weighed and measured then euthanized in 0.2% benzocaine, fixed measured then euthanized in 0.2% benzocaine, fixed in Bouin’s fixative, and preserved in 70% ethanol in Bouin’s fixative, and preserved in 70% ethanol until further analysis.until further analysis.


Tissues dissected and dehydrated in graded alcohols Tissues dissected and dehydrated in graded alcohols followed by infiltration with Histoclear and paraffinfollowed by infiltration with Histoclear and paraffin

Serial histological sections cut at 8µm through entire Serial histological sections cut at 8µm through entire gonadgonad

Slides were stained in Mallory’s trichrome stain and Slides were stained in Mallory’s trichrome stain and images of gonads were recordedimages of gonads were recorded

The collection sitesThe collection sites


Localities based on atrazine use determined by sales



Countries with <0.4kg/km² atrazine use were chosen as potential control sites




>9.3 kg/km² were chosen as potential atrazine-exposed sites




>9.3 kg/km² were chosen as potential atrazine-exposed sites

Collection sites continuedCollection sites continued


Sampled in Utah on 15 July 2001 and moved Sampled in Utah on 15 July 2001 and moved eastwardeastward



Collected 100 animals in 8 sites for a total of 800 Collected 100 animals in 8 sites for a total of 800 animalsanimals




Selected small individuals in an attempt to Selected small individuals in an attempt to sample newly metamorphosed animalssample newly metamorphosed animals





Animals were euthanized in benzocaine, fixed in Animals were euthanized in benzocaine, fixed in Bouin’s fixative for 48 hours and preserved in Bouin’s fixative for 48 hours and preserved in 70% ethanol70% ethanol





Animals were euthanized in benzocaine, fixed in Animals were euthanized in benzocaine, fixed in Bouin’s fixative for 48 hours and preserved in Bouin’s fixative for 48 hours and preserved in 70% ethanol70% ethanol

They were measured and sex was determined They were measured and sex was determined and histological analysis was conducted on the and histological analysis was conducted on the gonads of 20 males from each site and a subset gonads of 20 males from each site and a subset of females from each siteof females from each site

Chemical AnalysisChemical Analysis


At each site, 100mL of water was collectedAt each site, 100mL of water was collected


At each site, 100mL of water was collectedAt each site, 100mL of water was collected Frozen on dry ice Frozen on dry ice


At each site, 100mL of water was collectedAt each site, 100mL of water was collected Frozen on dry ice Frozen on dry ice Atrazine levels were determinedAtrazine levels were determined


At each site, 100mL of water was collectedAt each site, 100mL of water was collected Frozen on dry ice Frozen on dry ice Atrazine levels were determinedAtrazine levels were determined Water sample extracted in organic solvent Water sample extracted in organic solvent

followed by aqueous/organic extractionfollowed by aqueous/organic extraction



followed by aqueous/organic extractionfollowed by aqueous/organic extraction Analyzed by liquid chromatography/mass Analyzed by liquid chromatography/mass

spectrophotometry using daughter ionspectrophotometry using daughter ion




spectrophotometry using daughter ionspectrophotometry using daughter ion Positive controls contained mixtures of pesticides Positive controls contained mixtures of pesticides

at both 0.1 and 10.0 ppb at both 0.1 and 10.0 ppb




spectrophotometry using daughter ionspectrophotometry using daughter ion Positive controls contained mixtures of pesticides Positive controls contained mixtures of pesticides

at both 0.1 and 10.0 ppb at both 0.1 and 10.0 ppb detection limits at 0.1 ppbdetection limits at 0.1 ppb

Gonadal analysis on lab-animalsGonadal analysis on lab-animals


Control animals sexually differentiated at metamorphosis


Control animals sexually differentiated at metamorphosis earliest males to

metamorphose had solid testicular lobules




testes were more testes were more differentiated differentiated (contained more (contained more distinct tubules and distinct tubules and germ cells) anteriorly germ cells) anteriorly than posteriorlythan posteriorly




testes were more testes were more differentiated differentiated (contained more (contained more distinct tubules and distinct tubules and germ cells) anteriorly germ cells) anteriorly than posteriorlythan posteriorly

Gonadal analysis continuedGonadal analysis continued


Animals that Animals that metamorphosed later had metamorphosed later had open lobulesopen lobules


Animals that Animals that metamorphosed later had metamorphosed later had open lobulesopen lobules Contained both primary Contained both primary

spermatogonia and spermatogonia and spermatidsspermatids




Females had numerous Females had numerous oocytes in their gonads oocytes in their gonads and a central ovarian and a central ovarian cavitycavity




Females had numerous Females had numerous oocytes in their gonads oocytes in their gonads and a central ovarian and a central ovarian cavitycavity

The ResultsThe Results


Atrazine-treated males (0.1 Atrazine-treated males (0.1 and 25 ppb) were sexually and 25 ppb) were sexually differentiated at differentiated at metamorphosismetamorphosis


Atrazine-treated males (0.1 Atrazine-treated males (0.1 and 25 ppb) were sexually and 25 ppb) were sexually differentiated at differentiated at metamorphosismetamorphosis 36 and 12% of males 36 and 12% of males

treated with 0.1 and 25 treated with 0.1 and 25 ppb atrazine suffered ppb atrazine suffered gonadal dysgenesis gonadal dysgenesis


Atrazine-treated males (0.1 and Atrazine-treated males (0.1 and 25 ppb) were sexually 25 ppb) were sexually differentiated at metamorphosisdifferentiated at metamorphosis 36 and 12% of males treated 36 and 12% of males treated

with 0.1 and 25 ppb atrazine with 0.1 and 25 ppb atrazine suffered gonadal dysgenesis suffered gonadal dysgenesis

Gonadal dysgenesis- Gonadal dysgenesis- underdeveloped testes underdeveloped testes with poorly structured, with poorly structured, closed lobules or no closed lobules or no lobules at all and low to lobules at all and low to absent germ cellsabsent germ cells


Atrazine-treated males (0.1 and Atrazine-treated males (0.1 and 25 ppb) were sexually 25 ppb) were sexually differentiated at metamorphosisdifferentiated at metamorphosis 36 and 12% of males treated 36 and 12% of males treated

with 0.1 and 25 ppb atrazine with 0.1 and 25 ppb atrazine suffered gonadal dysgenesis suffered gonadal dysgenesis

Gonadal dysgenesis- Gonadal dysgenesis- underdeveloped testes underdeveloped testes with poorly structured, with poorly structured, closed lobules or no closed lobules or no lobules at all and low to lobules at all and low to absent germ cellsabsent germ cells

The Results ContinuedThe Results Continued


29% of the 0.1 ppb-treated animals and 8% that 29% of the 0.1 ppb-treated animals and 8% that were treated with 25 ppb displayed varying were treated with 25 ppb displayed varying degrees of sex reversaldegrees of sex reversal



Testicular lobules of sex-reversed males Testicular lobules of sex-reversed males contained oocytescontained oocytes




Males that metamorphosed later contained large Males that metamorphosed later contained large number of oocytesnumber of oocytes





Males that underwent complete sex reversal had Males that underwent complete sex reversal had gonads almost completely filled with oocytes and gonads almost completely filled with oocytes and only a limited number of lobules remained. only a limited number of lobules remained.





Males that underwent complete sex reversal had Males that underwent complete sex reversal had gonads almost completely filled with oocytes and gonads almost completely filled with oocytes and only a limited number of lobules remained. only a limited number of lobules remained.

No observable effects in atrazine-treated femalesNo observable effects in atrazine-treated females

Observations in the WildObservations in the Wild


Chemical analysis of water revealed none of the Chemical analysis of water revealed none of the sites were atrazine free sites were atrazine free


Chemical analysis of water revealed none of the Chemical analysis of water revealed none of the sites were atrazine free sites were atrazine free Only one site (Juab County, Utah) contained Only one site (Juab County, Utah) contained

<0.2 ppb atrazine levels<0.2 ppb atrazine levels



<0.2 ppb atrazine levels<0.2 ppb atrazine levels Sites in Utah and Wyoming did not have Sites in Utah and Wyoming did not have

detectable levels of atrazine metabolitesdetectable levels of atrazine metabolites



<0.2 ppb atrazine levels<0.2 ppb atrazine levels Sites in Utah and Wyoming did not have Sites in Utah and Wyoming did not have

detectable levels of atrazine metabolitesdetectable levels of atrazine metabolites All sites with atrazine sales exceeding 0.4 kg/km² All sites with atrazine sales exceeding 0.4 kg/km²

and contaminant levels that exceeded 0.2 ppb and contaminant levels that exceeded 0.2 ppb had males that displayed sex reversal similar to had males that displayed sex reversal similar to those that resulted from laboratory those that resulted from laboratory

Observations in the Wild Observations in the Wild ContinuedContinued


Site 3, North Platte Site 3, North Platte River in Wyoming had River in Wyoming had most advanced cases most advanced cases of hermaphroditism of hermaphroditism


Site 3, North Platte Site 3, North Platte River in Wyoming had River in Wyoming had most advanced cases most advanced cases of hermaphroditism of hermaphroditism 92% testicular 92% testicular

oocytesoocytes


Site 3, North Platte Site 3, North Platte River in Wyoming had River in Wyoming had most advanced cases most advanced cases of hermaphroditism of hermaphroditism 92% testicular 92% testicular

oocytesoocytes Advanced stages of Advanced stages of

complete sex complete sex reversalreversal


Site 3, North Platte Site 3, North Platte River in Wyoming had River in Wyoming had most advanced cases of most advanced cases of hermaphroditism hermaphroditism 92% testicular 92% testicular


complete sex reversalcomplete sex reversal All other sites varied in All other sites varied in

frequency and severity frequency and severity of gonadal of gonadal abnormalitiesabnormalities


Site 3, North Platte Site 3, North Platte River in Wyoming had River in Wyoming had most advanced cases of most advanced cases of hermaphroditism hermaphroditism 92% testicular 92% testicular


complete sex reversalcomplete sex reversal All other sites varied in All other sites varied in

frequency and severity frequency and severity of gonadal of gonadal abnormalitiesabnormalities

Contaminant levelsContaminant levels

Contaminant levelsContaminant levels

The Collection SitesThe Collection Sites

The Collection SitesThe Collection Sites

Contaminant levels continuedContaminant levels continued

Contaminant levels continuedContaminant levels continued

What does all this mean?What does all this mean?


Atrazine exposure disrupts gonadal development Atrazine exposure disrupts gonadal development in exposed larvaein exposed larvae



Widespread atrazine contamination was Widespread atrazine contamination was accompanied by observation of hermaphroditic accompanied by observation of hermaphroditic animals in the fieldanimals in the field




Studies suggest that atrazine impacts amphibians Studies suggest that atrazine impacts amphibians in the wildin the wild




Studies suggest that atrazine impacts amphibians Studies suggest that atrazine impacts amphibians in the wildin the wild Atrazine contamination of water coincides with Atrazine contamination of water coincides with

amphibian breeding activityamphibian breeding activity

What does this mean to us?What does this mean to us?


Not so much that amphibian population declines Not so much that amphibian population declines (decline is minimal)(decline is minimal)



We drink the same waterWe drink the same water



We drink the same waterWe drink the same water Atrazine effects on amphibians vs. atrazine Atrazine effects on amphibians vs. atrazine

effects on humanseffects on humans




effects on humanseffects on humans May cause reproduction problems if long term May cause reproduction problems if long term

exposure to atrazineexposure to atrazine




effects on humanseffects on humans May cause reproduction problems if long term May cause reproduction problems if long term

exposure to atrazineexposure to atrazine ““Adverse effects Adverse effects on hypothalamic-pituitary

function in humans”--Scientific Advisory Panel convened in June 2000

Risk and ExposureRisk and Exposure

Risk and ExposureRisk and Exposure

The Bigger PictureThe Bigger Picture


Why should we care?Why should we care?


Why should we care?Why should we care? Atrazine contamination may affect us alsoAtrazine contamination may affect us also


Why should we care?Why should we care? Atrazine contamination may affect us alsoAtrazine contamination may affect us also May not be same effects exhibited in May not be same effects exhibited in

amphibiansamphibians



amphibiansamphibians More aware of other pesticidesMore aware of other pesticides




How do we solve Atrazine Contamination?How do we solve Atrazine Contamination?




How do we solve Atrazine Contamination?How do we solve Atrazine Contamination? Prohibit atrazine distributionProhibit atrazine distribution




How do we solve Atrazine Contamination?How do we solve Atrazine Contamination? Prohibit atrazine distributionProhibit atrazine distribution Come up with new pesticide Come up with new pesticide

SourcesSources

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460 http://www.epa.gov/oppsrrd1/reregistration/atrazine/hed_redchap_16apr02.PDF

Atrazine-Induced Hermaphroditism at 0.1 ppb in American Leopard Frogs (Rana pipiens): Laboratory and Field Evidence http://ehp.niehs.nih.gov/members/2003/5932/5932.pdf

Feminized Frogs: Herbicide disrupts sexual growth http://www.sciencenews.org/articles/20020420/fob1.asp

Documents

Atrazine-Induced Hermaphroditism By: Michelle Lin